Trichomes are various outgrowths of the epidermis in plants including branched and unbranched hairs, vesicles, hooks spines and stinging hairs. Trichomes are staked, multicellular protruding structures that are considered important in the protection of plants against herbivores, but also against water loss by transpiration and UV irradiation (Mauricio & Rausher, 1997, Evolution 51, 1435 Wagner et al. 2004, Ann Bot 93, 3), as sinks for toxic heavy metals and xenobiotics (Salt et al., 1995, Plant Phys 109, 1427; Domínguez-Solís et al. 2001, J Biol Chem 276, 9297; Gutiérrez-Alcalá et al. 2000, PNAS 97, 11108).
Glandular trichomes containing various secondary compounds are present on the foliage of many solanaceous species and their role in resistance to various pests has been well documented. Glandular trichomes in tomato contain high amounts of terpenes (van der Hoeven et al., Plant Cell. 2000, vol. 12(11):2283-2294) and methylketones (Fridman et al., Plant Cell 2005, vol. 17(4):1252-1267). Trichomes have been categorised into types I to VII (Luckwill, 1943, Aberdeen University Press, UK) with types I, IV, VI and VII as glandular trichome types and II, III, and V as non-glandular. Type IV, V and VI are the prevalent trichomes on the wild tomato S. habrochaites (Simmons and Gurr, 2005, Agricultural and Forest Entomol 7, 265). In S. pennelli resistance to pests is predominantly related to the chemistry and density of type IV glandular trichomes, which cover all parts of the plant (Simmons et al., 2003 Aus J Entomol 43, 196; 2004, Entomol Exp App 113, 95). When exudates of glandular trichomes are physically removed, pest survival and longevity increases while mortality and entrapment decreases (Simmons and Gurr, 2005, Agricultural and Forest Entomol 7, 265). Trichome types IV and VI have been positively correlated with pest control.
Non-glandular trichomes of cotton ovules have been explored as targets for biotechnology with high economic importance (Kim and Triplett, 2001, Plant Physiol 127, 1361). The ability of glandular trichomes to secrete various phytochemicals, such as acyl sugars in S. pennelli and methyl ketones and terpenes in S. habrochaites, makes these structures potentially suitable for biotechnological application and provides opportunities for trichome based pest management.
Protein expression in trichomes can be utilised for the production of useful compounds such as pesticides, pharmaceutical components, volatile oils, flavours and fragrances (Callow, 2000 Advances in botanical research eds. Hallahan and Gray, San Diego Academic Press; Wagner 2004 Ann Bot 93, 3). In order to generate components specifically in trichomes, regulation of gene expression in the plant trichome is necessary. The possibility to direct protein expression in specialised structures or cells avoids interference in the plant metabolic pathways and consequently the performance of the plant.
Constitutive strong promoters such as the well known Cauliflower Mosaic Virus promoter (CaMV 35S) promoter are generally used in ectopic-expression studies. However this type of promoter is not very suitable for the expression of specific and possibly phytotoxic compounds, such as terpenes. Moreover, exhaustion of metabolic pools might be problematic.
Gutierrez-Alcala (2005, J Exp Bot 56, 2487) describes the promoter of the Arabidopsis thaliana OASA1 gene, which has activity in both glandular and non-glandular trichomes of tobacco.
Wang et al. (2002, J Exp Bot 53, 1891) describes a trichome specific promoter from tobacco P450 gene, CYP71D16, which shows expression in tobacco glandular trichomes at all developmental stages.
WO2004/111183 describes trichome specific promoters from tomato and tobacco leaves. However, upon testing transgenics described in WO2004/111183 no satisfactory results were obtained in the sense that expression was not trichome specific (e.g. additional expression in leaf veins) and expression was weak.
WO2009/082208 describes trichome-specific promoters from tomato. Although these promoters have been shown to be strictly trichome-specific, their activity is relatively low.
Despite trichome specific promoters having been isolated, none show a combination of trichome-specificity and high promoter activity. There is a clear need for highly active trichome-specific or trichome preferred promoters. Moreover, ideally several such promoters should be identified in order to successfully engineer the production of useful compounds in trichomes. Biosynthesis of these compounds often require the use of multiple genes, and thus ideally these multiple genes are expressed in trichomes, preferably using various promoters.
Such new plant promoters should ideally have the following requirements: i. organ/tissue specificity, the promoters should be strictly trichome-specific to avoid potentially unwanted compound production in other tissues (e.g. edible part); ii. high promoter activity.
The present invention provides trichome specific transcription regulatory sequences which are suitable for directing expression of operably linked nucleic acid molecules in glandular and non-glandular trichomes, especially in glandular trichomes found on various plant surfaces (aerial parts such as leaves, stems, floral organs) while, in some species, they are absent from particular plant surfaces such as the fruit and seeds. Simple trichomes are present on aerial surfaces of most angiosperms and some gymnosperms and bryophytes (Wagner et al. 2004, Ann Bot 93, 3). In angiosperms trichomes may occur on leaves, petals, petioles, peduncles, stems and seed coats, depending on the species. Glandular trichomes are found on perhaps 30% of vascular plants (Dell and McComb, 1978, Adv Bot Res 6, 227; Fahn, 2000 Adv Bot Res 31, 37).